Endocytosing delivery vehicle for targeting drugs and antibodies to prostate and other tissues
AssigneeThe Regents of the University of California
InventorAyyappan Rajasekaran
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Abstract
A chimeric transmembrane protein comprising a membrane-spanning polypeptide and an internalization motif of the sequence MXXXL, where X is any amino acid. The chimeric protein can be expressed on the surface of a cell and internalized. Polynucleotides and expression vectors encoding chimeric transmembrane proteins are also provided. Expression of chimeric proteins in the plasma membrane of target cells, followed by binding of antibodies to the chimeric proteins, where the antibodies are conjugated to a cytotoxic agent, provides a means to deliver the cytotoxic agent to the inside of the target cells.
Claims59
A chimeric transmembrane protein comprising:
- a membrane-spanning polypeptide;
- and
- an internalization motif of the sequence MX1X2X3L or LX3X2X1M connected to the membrane-spanning polypeptide;
- wherein at least a portion of the membrane-spanning polypeptide is heterologous to the internalization motif,
- X1, X2, and X3 are each independently any amino acid, provided that X1 is not tryptophan and X2 is not asparagine when X3 is leucine or alanine,
- the internalization motif occurs at a cytoplasmically located part of the chimeric transmembrane protein, and
- wherein the chimeric transmembrane protein is internalized when expressed in the plasma membrane of a eukaryotic cell.
A chimeric transmembrane protein comprising:
- a membrane-spanning polypeptide;
- and
- an internalization motif of the sequence MX1X2X3L or LX3X2X1M connected to the membrane-spanning polypeptide;
- wherein at least a portion of the membrane-spanning polypeptide is heterologous to the internalization motif,
- X1, X2, and X3 are each independently any amino acid, provided that X1 is not tryptophan and X2 is not asparagine when X3 is leucine or alanine,
- the internalization motif is located at the N- or C-terminus of the chimeric transmembrane protein, forming a cytoplasmic tail, and
- the chimeric transmembrane protein is internalized when expressed in the plasma membrane of a eukaryotic cell.
A method of modifying a first polypeptide for targeting to endosomes, comprising connecting the first polypeptide to a second polypeptide comprising an internalization motif of the sequence MX1X2X3L or LX3X2X1M such that a chimeric transmembrane protein is formed, the chimeric transmembrane protein comprising
- a membrane-spanning polypeptide;
- and
- the internalization motif;
- wherein at least a portion of the membrane-spanning polypeptide is heterologous to the internalization motif,
- X1, X2, and X3 are each independently any amino acid, provided that X1 is not tryptophan and X2 is not asparagine when X3 is leucine or alanine,
- the internalization motif occurs at a cytoplasmically located part of the chimeric transmembrane protein, and
- the chimeric transmembrane protein is internalized when expressed in the plasma membrane of a eukaryotic cell.
A method of modifying a first polypeptide for targeting to endosomes, comprising connecting the first polypeptide to a second polypeptide comprising an internalization motif of the sequence MX1X2X3L or LX3X2X1M such that a chimeric transmembrane protein is formed, the chimeric transmembrane protein comprising
- a membrane-spanning polypeptide;
- and
- the internalization motif;
- wherein at least a portion of the membrane-spanning polypeptide is heterologous to the internalization motif,
- X1, X2, and X3 are each independently any amino acid, provided that X1 is not tryptophan and X2 is not asparagine when X3 is leucine or alanine,
- the internalization motif is located at the N- or C-terminus of the chimeric transmembrane protein, forming a cytoplasmic tail, and
- the chimeric transmembrane protein is internalized when expressed in the plasma membrane of a eukaryotic cell.
The method of claim 28, wherein the membrane-spanning polypeptide has an amino acid sequence obtained or derived from the α-chain of the interleukin-2 receptor, and the internalization motif has the sequence LX3X2X1M which is located at the C-terminus of the chimeric transmembrane protein.
A method of providing a cytotoxic molecule to a cell, comprising:
- a) expressing a chimeric transmembrane protein in the plasma membrane of the cell, the chimeric transmembrane protein comprising
- a membrane-spanning polypeptide, and
- an internalization motif of the sequence MX1X2X3L or LX3X2X1M connected to the membrane-spanning polypeptide,
- wherein at least a portion of the membrane-spanning polypeptide is heterologous to the internalization motif,
- X1, X2, and X3 are each independently any amino acid, provided that X1 is not tryptophan and X2 is not asparagine when X3 is leucine or alanine,
- the internalization motif occurs at a cytoplasmically located part of the chimeric transmembrane protein, and
- the chimeric transmembrane protein is internalized following expression in the plasma membrane;
- and
- b) administering an antibody conjugated to the cytotoxic molecule, said antibody recognizing an extracellular portion of the membrane-spanning polypeptide;
- wherein the antibody-conjugate is internalized upon binding to the chimeric transmembrane protein.
Dependencies are inferred from the claim text.